This proposal is a renewal of 5R01EY013010-12 Exclusion & Coordination of rh genes. Visual information is gathered in the Drosophila compound eye by photoreceptors specialized in various tasks. We focus on the different types of photoreceptors involved in color vision. They express Rhodopsin photopigments that detect lights of different wavelengths: R7 photoreceptors express a UV sensitive Rhodopsin (Rh3 or Rh4), and R8 express either a Blue (Rh5) or a Green (Rh6) Rhodopsin. Color vision is achieved through comparison of light information received by the R7 and R8 of each individual eye (ommatidia). Since there are two types of ommatidia, Rh3 input is compared with Rh5, or Rh4 with Rh6. The two types of ommatidia are distributed stochastically in the retina with a 30:70 ratio. Their specification is controlled by the transcription factor Spineless that is it expressed stochastically in a subset of R7 cells. In aim 1, we propose to investigate the molecular mechanisms that control how spineless is intrinsically expressed in a stochastic manner. We will identify the different elements of the spineless promoters that control expression in all R7 cells, and those that lead to repression in a stochastic subset. We will use biochemical approaches such as 3C and Chromatin ImmunoPrecipitation to investigate the molecular mechanisms involved in the repression. We will also take advantage of natural variants mapping at spineless that have dramatically decreased ratio of Rh3:Rh4 to define the elements that control this ratio. In aim 2, we will generalize these observations to define the mechanisms by which the rhodopsin promoters integrate the information about cell specification to drive high level and yet exquisitely specific expression of different rhodopsins in distinct photoreceptor types that are otherwise highly related. We will investigate how elements common to all rhodopsin genes drive broad photoreceptor expression while specific elements restrict expression to their appropriate subset. We will identify cis-acting elements as well as the transcription factors that act on these sites. We will then use our deep knowledge of the system to reconstruct the rhodopsin promoters with defined elements reassembled as synthetic promoters. This achievement would represent a unique example where we understand the grammar of the transcription code and would be applicable to elucidating mechanisms of control of other terminal differentiation genes such as vertebrate opsins. PUBLIC HEALTH RELEVANCE: Drosophila, with its genetic amenability and its sophisticated visual behavior, has been very successfully developed as a model system to study retinal patterning. We investigate how the different subtypes of photoreceptor neurons are generated and distributed in the retina to achieve motion detection and color vision, in particular, studying the mechanisms by which stochastic choices increase diversity of photoreceptors and how the promoters of rhodopsin genes are regulated. The principles deduced from this project will be applicable to our understanding of patterning of the human retina.